Phage for lysing drug-resistant staphylococcus aureus and application thereof

Abstract

The application discloses a bacteriophage for lysing drug-resistant staphylococcus aureus and an application thereof. The bacteriophage is isolated from sewage and can lyse drug-resistant staphylococcus aureus, and the optimal MOI range of the bacteriophage for drug-resistant staphylococcus aureus is 0.03-0.27. The bacteriophage can inhibit the growth of drug-resistant staphylococcus aureus strains from dermatitis in different MOI ranges, has a good bacteriostatic effect, and provides a new and more effective selection for treating dermatitis caused by drug-resistant staphylococcus aureus strains.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparations, specifically relating to a bacteriophage for lysing drug-resistant Staphylococcus aureus and its application. Background Technology

[0002] Staphylococcus aureus (S. aureus), also known as "Staphylococcus aureus," belongs to the genus Staphylococcus and is a representative Gram-positive bacterium. It is a common foodborne pathogenic microorganism closely related to the occurrence and aggravation of dermatitis. The colonization rate of Staphylococcus aureus in the skin of healthy individuals is approximately 5-30%, while in patients with skin lesions, the colonization rate can reach 75-100%, and even in non-lesional patients, it can reach at least 30%. Clinically, antibiotics are commonly used to combat bacterial infections. However, with the large-scale production and use of antibiotics, especially their overuse, a large number of drug-resistant bacteria have emerged, ultimately leading to the emergence of multidrug-resistant (MDR) bacteria and ESKAPE super-resistant bacteria.

[0003] Bacteriophages are viruses that infect bacteria. Due to their specific ability to kill pathogens, low toxicity, and lack of disruption to the body's normal microbiota and the absence of severe endotoxemia, they are considered a promising alternative to antibiotics and have regained researchers' attention. Patent CN109082414A discloses a Staphylococcus aureus phage that exhibits good in vitro and in vivo antibacterial activity against drug-resistant Staphylococcus aureus. However, because it cannot inhibit Staphylococcus aureus at a MOI of 1, and only at a MOI of 10 can it effectively treat skin infections caused by Staphylococcus aureus, and generally, cells with higher MOI values ​​are less susceptible to infection, the phage's effect on inhibiting drug-resistant Staphylococcus aureus is not ideal. Therefore, it is necessary to obtain a phage with better inhibitory effects on drug-resistant Staphylococcus aureus for the treatment of skin infections. Summary of the Invention

[0004] The purpose of this invention is to provide a bacteriophage for lysing Staphylococcus aureus, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M2023496.

[0005] Furthermore, the phage is a phage that lyses drug-resistant Staphylococcus aureus.

[0006] Furthermore, the phage is a phage of drug-resistant Staphylococcus aureus that lyses the dermatitis-causing bacteria.

[0007] Furthermore, the drug-resistant Staphylococcus aureus is a Staphylococcus aureus resistant to chloramphenicol, lincomycin, erythromycin, kanamycin, ampicillin, and / or cefuroxime axetine.

[0008] The present invention also provides the use of the aforementioned bacteriophage in the preparation of an antibacterial agent that inhibits Staphylococcus aureus.

[0009] Furthermore, the antibacterial agent is an antibacterial agent that inhibits drug-resistant Staphylococcus aureus.

[0010] Furthermore, the antibacterial agent is an antibacterial agent that inhibits drug-resistant Staphylococcus aureus originating from dermatitis; the drug-resistant Staphylococcus aureus is Staphylococcus aureus resistant to chloramphenicol, lincomycin, erythromycin, kanamycin, ampicillin and / or cefuroxime axetil.

[0011] Finally, this invention provides the use of the aforementioned bacteriophage in the preparation of an antibacterial drug, wherein the drug is an anti-Staphylococcus aureus drug.

[0012] Furthermore, the drug is an antidote for drug-resistant Staphylococcus aureus.

[0013] Furthermore, the drug has the effect of inhibiting the growth of drug-resistant Staphylococcus aureus originating from dermatitis; the drug-resistant Staphylococcus aureus is Staphylococcus aureus resistant to chloramphenicol, lincomycin, erythromycin, kanamycin, ampicillin and / or cefuroxime axetine.

[0014] This invention relates to Staphylococcus aureus bacteriophage VB. _ SauM_SI2(Staphylococcus aureusbacteriophage VB _ SauM_SI2 was deposited at the China Center for Type Culture Collection (CCTCC) on April 10, 2023, with accession number CCTCC NO:M 2023496. The depositary address is: Wuhan University, Wuhan, China, 430072, China.

[0015] This invention relates to a Staphylococcus aureus bacteriophage, a lytic bacteriophage isolated from wastewater using Staphylococcus aureus strain as the host. This bacteriophage can lyse the host bacteria and form clear, transparent plaques with neat edges and a diameter of approximately 1.65±0.24 mm on a double-layer plate. Electron microscopy results show that its head diameter is 96±3.9 nm, neck length is 16.4±1.4 nm, tail length is 110±4.4 nm, diameter is 27±0.1 nm, and tail tube length is 104±5.6 nm. The nucleic acid chain type is dsDNA, belonging to Ca2+. Myoviridae (family Myoviridae) under the order Udovirales; its main structural protein has a molecular weight of approximately 55 kDa, exhibits stable infectious activity for up to 60 minutes below 50°C, and is stable for up to 60 minutes within a pH range of 5.0 ± 2.0; its optimal MOI range for host bacteria is 0.03-0.27; it can inhibit the growth of drug-resistant Staphylococcus aureus strains causing dermatitis within different MOI ranges, demonstrating good antibacterial effects and providing a new and more effective option for the treatment of dermatitis caused by drug-resistant Staphylococcus aureus strains.

[0016] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0017] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0018] Figure 1 Genome images by 1% agarose gel electrophoresis (a: partial genome of the tested bacteria, lane M: DL5000 bp DNA marker, lanes 1-2: two of the tested strains; b: 16S amplification results of some of the tested strains, M: DL5000 DNA marker, lanes 1-8: eight of the tested strains)

[0019] Figure 2 Evolutionary tree diagram of test strains and reference strains

[0020] Figure 3 1% agarose gel electrophoresis image of spa typing PCR amplification products (lane M: DL5000 DNA Marker; lanes 1-9: PCR amplification products of 9 strains of Staphylococcus aureus from dermatitis).

[0021] Figure 4 Plaques and morphology of bacteriophage VB_SauM_SI2 (a: plaques of bacteriophage on double-layer plates; b: morphology of bacteriophage under transmission electron microscopy)

[0022] Figure 5 Genomic images and 1% agarose gel electrophoresis images after enzyme digestion (lane 1 is for phage VB). _ SauM_SI2; a: Genome; Lane M: DL5,000 DNA marker; b: DNase I digestion product; Lane M: DL7,000 DNA marker; c: RNase A digestion product; Lane M: DL15,000 DNA marker; d: Exonuclease I digestion product; Lane M: DL5,000 DNA marker)

[0023] Figure 6 Phage VB_SauM_SI2 phylogenetic tree

[0024] Figure 7 Bacteriophage VB _ SDS-PAGE analysis of SauM_SI2 structural protein

[0025] Figure 8 Bacteriophage VB _ SauM_SI2 stability curve (a: thermal stability. Within 60 min, phage VB...) _ The titer variation of SauM_SI2 at different temperatures, with the ordinate representing the mean ± standard deviation (SD) of the titer; b: pH stability; within 60 min, phage VB _ The titer variation of SauM_SI2 at different pH values ​​is shown on the ordinate (mean ± standard deviation SD).

[0026] Figure 9 Bacteriophage VB _ SauM_SI2's antibacterial effect against 10 strains of Staphylococcus aureus Detailed Implementation

[0027] Example 1: Study on the antibacterial effect of Staphylococcus aureus bacteriophage VB_SauM_SI2 against drug-resistant Staphylococcus aureus strains originating from dermatitis.

[0028] 1. Materials and Methods

[0029] 1.1 Strains and Sample Sources

[0030] Strains were sourced from nine Staphylococcus aureus strains from dermatitis patients donated by the National Center for the Preservation of New Antibiotic Strains (numbered SA1, SA5, SA6, SA7, SA8, SA18, SA24, SA38, and SA39, respectively); and one non-clinical Staphylococcus aureus strain, SIIA1005, donated by the National Center for the Preservation of New Antibiotic Strains.

[0031] Wastewater source and pretreatment: Samples were taken from the wastewater system of a research institute in Chengdu. Large impurities were removed by centrifugation at 10,000 r / min for 2 min. The supernatant was filtered through a 0.45 μm filter membrane for sterilization, and the filtrate was stored at 4℃ for later use.

[0032] 1.2 Identification of Staphylococcus aureus 16S

[0033] 1.2.1 Strain amplification and genome extraction

[0034] Ten Staphylococcus aureus strains preserved with 20% glycerol were streaked onto LB agar plates and incubated upside down at 37°C for 18-24 hours. Single colonies were picked and inoculated into 3 mL of LB broth and cultured at 37°C with shaking at 220 rpm for 18 hours.

[0035] Genomic DNA was extracted according to the instructions of the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit. The concentration of the nucleic acid solution was detected using a microplate reader and stored at -20°C. The quality of the genome was verified by 1% agarose gel electrophoresis.

[0036] 1.2.2 PCR Amplification and Sequencing

[0037] Using the extracted genome as a template, PCR amplification was performed using the universal 16S primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′). PCR reaction conditions: 98℃ for 5 min; (98℃ for 30 s, 53℃ for 30 s, 72℃ for 2 min) 30 cycles; 72℃ for 5 min. PCR mixing system (50 μL): 20 μL Gold Mix (green), 1 μL 27F (10 μM), 1 μL 1492R (10 μM), 50-500 ng DNA template, and ultrapure water to make up to 50 μL. The size of the PCR amplification product bands was verified by 1% agarose gel electrophoresis. The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. (Chengdu Branch) for sequencing. The sequencing results were imported into the NCBI website for BLAST nucleic acid alignment. Multiple sequence nucleic acid alignment was performed using MEGAX software, and a phylogenetic tree was constructed using the MLE method.

[0038] 1.3 Staphylococcus aureus strain SPA typing

[0039] Using the extracted Staphylococcus aureus genome as a template, the variable repeat region (X region) of the spa in the genome was amplified using primers spa-1113F: 5′-TAAAGACGATCCTTCGGTGAGC-3′ and spa-1514R: 5′-CAGCAGTAGTGCCGTTTGCTT-3′. The PCR amplification products were analyzed by 1% agarose gel electrophoresis. The amplification products were sent to Beijing Qingke Biotechnology Co., Ltd. (Chengdu Branch) for sequencing and analyzed using Ridom software to determine the spa type of the strain. Staphylococcus aureus that could not be classified into any known spa type was considered untyped (NT).

[0040] PCR reaction conditions: 98℃ for 2 min; (98℃ for 25 s, 55℃ for 40 s, 72℃ for 60 s) 30 cycles, 72℃ for 10 min. The PCR mixing system is the same as that for 16S amplification.

[0041] 1.4 Staphylococcus aureus resistance test

[0042] Antibiotic resistance testing was performed using the Kirby-Bauer method and microbroth dilution method recommended by the European Union Testing Standards (EUCAST). Following the 2020 CLSI susceptibility testing standard M100, susceptibility testing was conducted on susceptibility discs for 11 commonly used antibiotics: chloramphenicol, ciprofloxacin, tetracycline, ampicillin, cefoxitin, kanamycin, tobramycin, erythromycin, rifampin, imipenem, and lincomycin, against 9 clinically derived Staphylococcus aureus strains. The quality control strain was SIIA1005.

[0043] Breakpoint determination was performed with reference to the 2020 CLSI Antimicrobial Susceptibility Standard M100. The criteria for determining the diameter of the inhibition zone are shown in Table 1.

[0044] Table 1. Criteria for Judging the Diameter of the Inhibition Zone in the KB Method Antimicrobial Susceptibility Test

[0045]

[0046]

[0047] Based on the results of the Kirby-Bauer disk diffusion method for drug resistance testing, three antibiotics that at least one strain of bacteria was resistant to and three antibiotics that were sensitive to were randomly selected, with one additional β-lactam antibiotic: cefuroxime axetil. The micro-broth dilution method was used for drug resistance testing, and this method was combined with the Kirby-Bauer method for drug resistance verification.

[0048] 1.5 Phage Isolation and Purification

[0049] 1.5.1 Phage Isolation

[0050] Using the non-clinical Staphylococcus aureus strain SIIA1005 as the host bacterium, bacteriophages were isolated from treated wastewater using the single-host enrichment method. 10 mL of the treated water sample and 1 mL of freshly prepared Staphylococcus aureus strain SIIA1005 suspension were added to a conical flask containing 10 mL of 2×LB broth. After mixing, the mixture was incubated at room temperature for 5 min, then at 37°C with shaking at 220 rpm for 10 h. After incubation, the culture was centrifuged at 12000 rpm for 10 min; the supernatant was collected and sterilized using a 0.22 μm filter membrane to obtain the first enrichment solution containing bacteriophages. The first enrichment solution was co-cultured with the bacterial suspension again for 10 h using the same method. This co-culture, centrifugation, and sterilization process was repeated twice to obtain the third enrichment solution containing bacteriophages. A Staphylococcus aureus culture without wastewater sample was used as a control.

[0051] The phage was validated using the spot method: freshly cultured SIIA1005 bacterial suspension was mixed with 3 mL of 0.6% LB agar and immediately poured onto a 1.5% LB lower agar plate. The mixture was allowed to stand at room temperature for 10 min and then incubated overnight at 30°C.

[0052] 1.5.2 Phage Purification

[0053] Phages were purified using double-layer agar plates (DLAP). A 10-fold serial dilution of phage enrichment solution was prepared by mixing 100 μL of the serially diluted enrichment solution with 100 μL of logarithmic growth phase Staphylococcus aureus suspension and then incubating at 30°C until plaques appeared.

[0054] Using a 1 mL sterile pipette tip, pick up a clear, bright, well-defined, and large single phage plaque into a 1.5 mL centrifuge tube containing 1 mL of SM buffer. Vortex for 1 min to dissolve the phage, centrifuge at 12000 rpm for 3 min, and use the supernatant for 10-fold serial dilution to obtain single phage plaques. Repeat this step until uniform phage plaques appear on the bilayer plate.

[0055] 1.5.3 Phage Amplification and Preservation

[0056] Select uniform phage plaques from the double-layer plate into 1 mL of SM buffer, centrifuge at 12000 r / min for 3 min, take 500 μL of supernatant, and use the enrichment method to co-culture with host bacteria to amplify the phage.

[0057] 1.5.4 PEG-concentrated phage particles

[0058] Phage concentration was performed using Pascale's PEG precipitation method. Specific steps:

[0059] 1) Add chloroform to a final concentration of 0.2% and incubate at 37°C for 30 minutes;

[0060] 2) Add to sodium chloride solution with a final concentration of 0.5M, and let stand at 4°C for at least 1 hour;

[0061] 3) Centrifuge the lysis buffer at 4°C, 8000×g, for 20 min to remove larger cell debris, and transfer the supernatant to a clean container;

[0062] 4) Add PEG8000 to a final concentration of 8-10%, mix well at 4°C, and let stand overnight;

[0063] 5) Centrifuge the overnight phage mixture at 4°C, 8000×g for 20 min, carefully remove the supernatant, and finally invert the centrifuge tube for 5 min;

[0064] 6) Resuspend the phage pellet in SM buffer (1 mL / 100 mL SM buffer);

[0065] 7) After resuspending, centrifuge at 4℃, 5000×g at low speed for 10 min;

[0066] 8) Extract with an equal volume of chloroform for 1 min, centrifuge at 5000×g for 15 min, and collect the aqueous phase containing the phage;

[0067] 9) Sterilize with a 0.22μm filter membrane to obtain a phage suspension.

[0068] 1.5.5 Phage Titer Determination

[0069] For each 10-fold serial dilution of phage SM buffer suspension, mix 100 μL of the phage suspension with 100 μL of logarithmic growth phase bacterial culture and incubate at room temperature for 10 min. Then, mix with 3 mL of 0.6% LB agar and immediately pour into a double-layer plate. Incubate inverted at 37°C overnight. Perform two replicates for each dilution. Select plates with 30-300 plaques for phage counting.

[0070] Titer (pfu / mL) = Average number of plaques (pfu) × Dilution factor 10 n / Amount added (mL)

[0071] 1.6 Transmission electron microscopy of bacteriophages

[0072] SM buffer phage suspension (10 11 The sample (pfu / mL) was sent to Chengdu Aochuang Biotechnology Co., Ltd. for transmission electron microscopy imaging.

[0073] 1.7 Phage nucleic acid chain verification

[0074] Take 5 mL of the amplified phage lysis buffer (titer ≥ 10). 9Genomic DNA was extracted using the HiPure Lambda Mini Kit (pfu / mL). DNase I and RNase A were used to verify that the nucleic acid type was DNA or RNA, respectively. Exonuclease I was used to verify that the phage nucleic acid strand type was dsDNA or ssDNA. All verifications were performed by nucleic acid electrophoresis on a 1% agarose gel.

[0075] 1.8 Verification of the molecular weight of major structural proteins of bacteriophage

[0076] Take the phage suspension purified by PEG (titer ≥10) 9 The sample was prepared by mixing the β-mercaptoethanol (pfu / mL) with 5× Loading buffer (containing 100 mM β-mercaptoethanol) and boiling at 100 °C for 10 min. SDS-PAGE electrophoresis was performed on a 12% separating gel. After staining and destaining, the gel block was photographed on a Biorad gel imaging system.

[0077] 1.9 Phage stability study

[0078] The temperature and pH stability of bacteriophages were investigated by referring to and modifying the method of Tan et al. Equal volumes of bacteriophage suspension were placed in constant temperature water baths at 40℃, 50℃, 60℃, and 70℃ for 1 h, respectively. The pH of LB broth was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 using 1M HCl and 1M NaOH, respectively. 5 μL of the bacteriophage suspension was mixed with 495 μL of LB broth at different pH values ​​and incubated at 37℃ for 1 h. Samples were taken at 0, 20, 40, and 60 min for each group, and the titer of the bacteriophages at each sampling point under different temperatures and pH values ​​was determined. Each treatment was repeated three times.

[0079] 1.10 Phage Host Range Determination

[0080] The lytic activity of the isolated bacteriophages against 10 strains of Staphylococcus aureus was determined using the dot bacteriophage assay to obtain the phage lysis profile (see 1.5.1 for the method). The presence or absence of phage plaques was observed; the formation of clear plaques indicated that the strain could be lysed.

[0081] 1.11 Determination of the antibacterial effect of bacteriophages against drug-resistant Staphylococcus aureus

[0082] The antibacterial activity of bacteriophages against host bacterium SIIA1005 and clinically derived drug-resistant Staphylococcus aureus was determined by slightly modifying the method of Benala et al.

[0083] Initial host bacteria adjusted to OD 600 ≈0.2 (approximately 1.0 × 10⁻⁶) 8 The initial phage titer was adjusted to 1.0 × 10⁻⁶ cfu / mL. 8Host bacteria and phages were obtained by serially diluting pfu / mL 10-fold. Equal volumes of phage and host bacteria at different concentrations were mixed to achieve MOIs of 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, and 1000. The effect of phages at each MOI on the growth of Staphylococcus aureus strains was determined in 96-well microplates to obtain the optimal MOI range for inhibiting Staphylococcus aureus growth. OD was measured using a Biotek microplate reader. 600 The value was measured every 60 minutes for a total of 360 minutes. Each treatment was repeated 3 times.

[0084] 2 Results

[0085] 2.1 Identification of 16S strains of Staphylococcus aureus

[0086] See (1% agarose gel electrophoresis images of nucleic acids of some tested strains) Figure 1 a). The gel image shows a distinct and specific amplified band at approximately 1500 bp, consistent with the expected size. Figure 1 b).

[0087] BLAST nucleic acid alignment results showed a similarity of over 99% with known Staphylococcus aureus strains. The phylogenetic tree constructed using the MLE method is shown below. Figure 2 The 10 test strains clustered in the same clade as the endogenous reference strain *S. aureus* ATCC 12600, but not in the same clade as the exogenous reference strains *S. epidermidis*, *S. lutrae*, *S. hominis*, *S. haemolyticus*, *S. cohnii*, and *S. saprophyticus*. This indicates that the test strains are more closely related to *S. aureus* in evolution. It can be confirmed that all 10 test strains belong to *S. aureus*.

[0088] 2.2 Staphylococcus aureus SPA typing

[0089] See gel electrophoresis of PCR amplification products. Figure 3 Spa typing results showed that 10 Staphylococcus aureus strains could be classified into 9 different spa types, and the spa type of 1 strain could not be determined (Table 2).

[0090] Table 2. Spa typing of Staphylococcus aureus strains

[0091]

[0092] 2.3 Staphylococcus aureus resistance test

[0093] The drug resistance results showed that each of the nine clinically derived Staphylococcus aureus strains was resistant to at least one antibiotic, while the quality control strain SIIA1005 was sensitive to all tested antibiotics. The drug resistance spectrum is shown in Table 3.

[0094] Five strains (56%) of *S. aureus* were resistant to at least two classes of antibiotics, and two strains (22%) were MDR strains (resistant to at least three antibiotics). The SA38 strain showed the highest co-resistance rate, exhibiting resistance to five antibiotics: chloramphenicol, lincomycin, erythromycin, kanamycin, and cefuroxime axetil. These results suggest that continued use of antibiotics to combat *S. aureus*-induced dermatitis will face increasing challenges; therefore, seeking new methods to combat multidrug-resistant *S. aureus* is urgently needed.

[0095] Table 3. Multidrug resistance spectrum of 10 Staphylococcus aureus strains

[0096]

[0097]

[0098] Note: "+" indicates drug resistance.

[0099] 2.4 Phage Isolation and Morphology

[0100] One bacteriophage strain was isolated and purified, named VB_SauM_SI2. On a host bacteriophage double-layer plate, the phage plaque diameter was approximately 1.65±0.24 mm, and the plaque was clear, translucent, and had neat edges. Figure 4 a).

[0101] Bacteriophages isolated from a single spot were analyzed by transmission electron microscopy (TEM). The phages belonged to the Myoviridae family within the order Caudovirales, possessing an icosahedral head and a retractable tail. The TEM image showed that phage VB_SauM_SI2 consisted of an icosahedral head with a diameter of 96±3.9 nm and a retractable tail with a length of 110±4.4 nm and a diameter of 27±0.1 nm. Figure 4 b), and has a clearly visible neck (b1), a base plate with a tail wire (b2), and a tail tube (b3).

[0102] Bacteriophage VB_SauM_SI2 was deposited at the China Center for Type Culture Collection (CCTCC) on April 10, 2023, with accession number CCTCC NO:M 2023496.

[0103] 2.5 Phage Nucleic Acid Types

[0104] The phage genome 1% agarose gel electrophoresis image showed a single band, and the quality was as expected. Figure 5 a). After DNase I digestion, the results showed that the genome was degraded (see...). Figure 5 b) RNase A digestion showed no degradation of the genome (see [link]). Figure 5c) This indicates that the phage's nucleic acid type is DNA; further digestion with Exonuclease I shows no degradation of the genome. Figure 5 d) indicates that the phage's nucleic acid is dsDNA. The fact that the phage's nucleic acid strand is dsDNA further proves that the phage belongs to Myoviridae (family Myoviridae) (nucleic acid strand type is dsDNA).

[0105] Genetic analysis of bacteriophage VB_SauM_SI2 and construction of a phylogenetic tree ( Figure 6 Among them, the similarity of bacteriophage VB_SauM_SI2 and Staphylococcus phage vB_SauH_SAP1, complete genome is 98%.

[0106] 2.6 Phage structural proteins

[0107] SDS-PAGE electrophoresis images ( Figure 7 Four protein bands, a, b, c, and d, are visible, with molecular weights of approximately 170, 80, 55, and 25 kDa, respectively. Among them, band c, with a molecular weight of approximately 55 kDa, has the highest content, suggesting that this protein is the main structural protein of the bacteriophage. This is close to the molecular weight of 42 kDa of the main structural protein of Staphylococcus aureus bacteriophage SA2 reported in the literature.

[0108] 2.7 Stability

[0109] Bacteriophage VB_SauM_SI2 exhibits high stability at temperatures below 50°C for at least 60 minutes, but its titer decreases to below the detection limit (1 lg pfu / mL) within 20 minutes at temperatures above 60°C. Figure 8 a).

[0110] The titer of bacteriophage VB_SauM_SI2 remained stable with no significant change in the pH range of 5.0-9.0, but decreased by 1-2 lg pfu / mL at pH 4.0 and pH 10.0-12.0, and dropped below the detection limit (1 lg pfu / mL) in the pH range of 2.0-3.0. Figure 8 b) This result indicates that bacteriophage VB_SauM_SI2 can remain stable and maintain its infectious activity in the pH range of 7.0±2.0. Its infectious activity is affected when the alkalinity is stronger (pH 10.0-12.0) and it almost loses its infectious activity when the acidity is stronger (pH 2.0-3.0).

[0111] 2.8 Phage Host Range

[0112] Host range assays showed that, in addition to the host bacterium SIIA1005, it exhibited strong lytic activity against seven other drug-resistant Staphylococcus aureus strains, and weaker lytic activity against the remaining two strains (SA39, SA39) (Table 4). Furthermore, all nine Staphylococcus aureus strains were drug-resistant, indicating that bacteriophage VB_SauM_SI2 has an activity against drug-resistant Staphylococcus aureus.

[0113] Table 4. Phage lysis range

[0114]

[0115] Note: "+" indicates that the plaque can be fragmented; the more plaques there are, the better the fragmentation effect (the clearer and brighter the plaques).

[0116] 2.9 Antibacterial effect of bacteriophage

[0117] The effects of phage VB_SauM_SI2 on the growth of host bacteria SIIA1005 and nine drug-resistant Staphylococcus aureus strains under different MOIs are shown in the figure. Figure 9 The optimal MOI range is summarized in Table 5. All control strains without phage addition were able to enter the logarithmic growth phase (OD). 600 >0.3), and its OD increases with time. 600 The value gradually increases; the increase in the proportion of bacteriophage added (MOI value) leads to an increase in the OD value of the strain growth. 600 The maximum value that can be achieved has decreased.

[0118] Within the optimal MOI range, phages can inhibit the entry of corresponding drug-resistant Staphylococcus aureus strains into the logarithmic growth phase (OD). 600 <0.3), and OD 600 The values ​​all showed a trend of first increasing and then decreasing. The optimal MOI ranges for drug-resistant Staphylococcus aureus strains SA38 and SA39 were 1.2-12 and 13.2-132, respectively, which were greater than the optimal MOI ranges for the other tested Staphylococcus aureus strains. This is consistent with the previous results in the lysis spectrum determination that the phage had a weaker lysis effect on these two Staphylococcus aureus strains than on other tested Staphylococcus aureus strains.

[0119] Table 5. Optimal MOI range for bacteriophages against Staphylococcus aureus strains

[0120]

[0121] 3. Discussion

[0122] This invention isolated a bacteriophage, VB_SauM_SI2, from environmental water samples using a non-clinically sourced (drug-resistant) Staphylococcus aureus strain from a bacterial bank as the host. The host range of this bacteriophage against drug-resistant (n=7) and multidrug-resistant Staphylococcus aureus strains (n=2) originating from dermatitis was determined. The results showed that the bacteriophage could lyse drug-resistant Staphylococcus aureus to varying degrees. Determining the host range is considered fundamental research before bacteriophage administration and plays an important guiding role in its therapeutic efficacy. Transmission electron microscopy results showed that it belongs to the Myoviridae family (Caudovirales) within the order Caudovirales. The bacteriophage's nucleic acid strand type is dsDNA, which, along with the morphological findings under electron microscopy, confirms its classification.

[0123] Investigating the stability of isolated bacteriophages under different temperature and pH conditions can provide guidance for the production, transportation, storage, and application of bacteriophages. Bacteriophage VB_SauM_SI2 maintains its infectious activity at temperatures below 50°C for 60 minutes. The production temperature should be within this range. This temperature also ensures that the bacteriophage preparation maintains its infectious activity during transportation under non-cold chain conditions. Furthermore, when applied to the skin surface, the temperature of the skin surface does not significantly affect its infectious activity. Bacteriophage VB_SauM_SI2 maintains its infectious activity within a pH range of 7.0 ± 2.0 for 60 minutes. Studies have shown that the pH of normal human skin surface is often acidic (pH 5.4-5.9), while the pH of dermatitis-affected skin is close to 7.0. The pH stability test results indicate that bacteriophage VB_SauM_SI2 can adapt to the pH of dermatitis-affected skin and maintain its lytic activity, providing a reference for the application of bacteriophages.

[0124] Furthermore, the bacteriophage VB_SauM_SI2 can infect and inhibit host Staphylococcus aureus and drug-resistant Staphylococcus aureus strains from dermatitis into the logarithmic growth phase at different optimal MOI ranges, indicating that this bacteriophage has the potential to treat dermatitis caused by drug-resistant Staphylococcus aureus.

Claims

1. A Staphylococcus aureus bacteriophage ( Staphylococcus aureus bacteriophage), characterized by: The phage is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2023496; The bacteriophage can lyse drug-resistant Staphylococcus aureus originating from dermatitis; The drug-resistant Staphylococcus aureus is a Staphylococcus aureus resistant to chloramphenicol, lincomycin, erythromycin, kanamycin, ampicillin, and / or cefuroxime axetine.

2. The use of the bacteriophage according to claim 1 in the preparation of an antibacterial agent for inhibiting drug-resistant Staphylococcus aureus originating from dermatitis, characterized in that: The drug-resistant Staphylococcus aureus is a Staphylococcus aureus resistant to chloramphenicol, lincomycin, erythromycin, kanamycin, ampicillin, and / or cefuroxime axetine.

3. The use of the bacteriophage according to claim 1 in the preparation of antibacterial drugs, characterized in that: The drug is an antidote for drug-resistant Staphylococcus aureus; The drug has the effect of inhibiting the growth of drug-resistant Staphylococcus aureus originating from dermatitis; the drug-resistant Staphylococcus aureus is Staphylococcus aureus resistant to chloramphenicol, lincomycin, erythromycin, kanamycin, ampicillin and / or cefuroxime axetine.

Images